Antibodies to liver derived receptors for advanced glycosylation endproducts and uses therefor

ABSTRACT

The present invention relates to receptors for advanced glycosylation endproducts derived from rat liver membranes, and that specifically comprise proteins determined to possess molecular masses of about 90 kD and 60 kD, respectively, as assessed by migration during SDS-PAGE. Partial N-terminal sequences have been determined and diagnostic and therapeutic agents, compositions and methods are proposed. Antibodies to the 90 kD and 60 kD receptor proteins are disclosed.

This invention was made with partial assistance from grant Nos. AG 8245and DK 19655 from the National Institutes of Health. The government mayhave certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Division of application Ser. No. 07/749,438 filedAug. 23, 1991 now U.S. Pat. No. 5,585,344, which is aContinuation-In-Part of application Ser. No. 453,958, filed Dec. 20,1989 now abandoned, which is in turn, a Division of application Ser. No.091,534, filed Sep. 3, 1987, now U.S. Pat. No. 4,900,747, issued Feb.13, 1990, which is in turn, a Continuation-In-Part of application Ser.No. 907,747, filed Sep. 12, 1986, now abandoned; all of the abovepreceding applications by Helen Vlassara, Michael Brownlee and AnthonyCerami, said Ser. No. 907,747, in turn, a Continuation-In-Part ofapplication Ser. No. 798,032, filed Nov. 14, 1985, by Anthony Cerami,Peter Ulrich and Michael Brownlee, now U.S. Pat. No. 4,758,583, whichis, in turn, a Continuation-In-Part of application Ser. No. 590,820, nowU.S. Pat. No. 4,665,192, filed Mar. 19, 1984 by Anthony Cerami alone.

Priority under 35 U.S.C. §120 is claimed as to all of the above earlierfiled Applications, and the disclosures thereof are incorporated hereinby reference.

RELATED PUBLICATIONS

The Applicants are co-authors of the following articles directed to thesubject matter of the present invention: "FUNCTION OF MACROPHAGERECEPTOR FOR NONENZYMATICALLY GLYCOSYLATED PROTEINS IS MODULATED BYINSULIN LEVELS", Vlassara, Brownlee and Cerami, DIABETES (1986), Vol. 35Supp. 1, Page 13a; "ACCUMULATION OF DIABETIC RAT PERIPHERAL NERVE MYELINBY MACROPHAGES INCREASES WITH THE PRESENCE OF ADVANCED GLYCOSYLATIONENDPRODUCTS", Vlassara, H., Brownlee, M., and Cerami, A. J. EXP. MED.(1984), Vol. 160, pp. 197-207; "RECOGNITION AND UPTAKE OF HUMAN DIABETICPERIPHERAL NERVE MYELIN BY MACROPHAGES", Vlassara, H., Brownlee, M., andCerami, A. DIABETES (1985), Vol. 34, No. 6, pp. 553-557;"HIGH-AFFINITY-RECEPTOR-MEDIATED UPTAKE AND DEGRADATION OFGLUCOSE-MODIFIED PROTEINS: A POTENTIAL MECHANISM FOR THE REMOVAL OFSENESCENT MACROMOLECULES", Vlassara H., Brownlee, M., and Cerami, A.,PROC. NATL. ACAD. SCI. U.S.A. (September 1985), Vol. 82, pp. 5588-5592;"NOVEL MACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS IS DISTINCTFROM PREVIOUSLY DESCRIBED SCAVENGER RECEPTORS", Vlassara, H., Brownlee,M., and Cerami, A. JOUR. EXP. MED. (1986), Vol. 164, pp. 1301-1309;"ROLE OF NONENZYMATIC GLYCOSYLATION IN ATHEROGENESIS", Cerami, A.,Vlassara, H., and Brownlee, M., JOURNAL OF CELLULAR BIOCHEMISTRY (1986),Vol. 30, pp. 111-120; "CHARACTERIZATION OF A SOLUBILIZED CELL SURFACEBINDING PROTEIN ON MACROPHAGES SPECIFIC FOR PROTEINS MODIFIEDNONENZYMATICALLY BY ADVANCED GLYCOSYLATION END PRODUCTS", Radoff, S.,Vlassara, H. and Cerami, A., ARCH. BIOCHEM. BIOPHYS (1988), Vol. 263,No. 2, pp. 418-423; "ISOLATION OF A SURFACE BINDING PROTEIN SPECIFIC FORADVANCED GLYCOSYLATION ENDPRODUCTS FROM THE MURINE MACROPHAGE-DERIVEDCELL LINE RAW 264.7", Radoff, S., Vlassara, H., and Cerami, A.,DIABETES, (1990), Vol. 39, pp. 1510-1518; "TWO NOVEL RAT LIVER MEMBRANEPROTEINS THAT BIND ADVANCED GLYCOSYLATION ENDPRODUCTS: RELATIONSHIP TOMACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS", Yang, Z., Makita,Z., Horii, Y., Brunelle, S., Cerami, A., Sehajpal, P., Suthanthiran, M.and Vlassara, H., J. EXP. MED., (1991), Vol. 174 pp. 515-524. All of theforegoing publications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the nonenzymaticglycosylation of proteins, and particularly to the discovery of bindingpartners to advanced glycosylation endproducts such as AGE receptors,that may serve in the diagnosis and treatment of conditions in which thepresence or activity of such advanced glycosylation endproducts may beimplicated.

Glucose and other reducing sugars attach non-enzymatically to the aminogroups of proteins in a concentration-dependent manner. Over time, theseinitial Amadori adducts undergo further rearrangements, dehydrations andcross-linking with other proteins to accumulate as a family of complexstructures which are referred to as Advanced Glycosylation Endproducts(AGEs). Although this chemistry has been studied by food chemists formany years, it was only in the past decade that the presence of AGEs inliving tissues has been established. The excessive deposition of theseproducts on structural body proteins as a function of age and elevatedglucose concentration, taken together with evidence of effectiveprevention of tissue pathology by an AGE inhibitor, aminoguanidine, haslent support to the hypothesis that the formation of AGEs plays a rolein the long term complications of aging and diabetes.

Since the amount of AGEs found in human tissues is less than could bepredicted from protein/glucose incubation studies in vitro, theapplicants herein proposed several years ago that there might be normalmechanisms to remove those long-lived proteins which had accumulatedAGEs in vivo. Particularly, and as set forth initially in parentapplication Ser. No. 907,747, now abandoned and the above-referencedapplications that have followed, monocytes/macrophages were found todisplay high affinity surface binding activity specific for AGEmoieties, independent of the protein which was AGE-modified. Thismacrophage AGE-receptor was shown to differ from other known scavengerreceptors on these cells. In addition, an endogenous means for the invivo elimination or removal of advanced glycosylation endproducts wasset forth, and corresponding diagnostic applications involving thereceptors and including a specific receptor assay were also proposed.

Following this determination, the applicants herein have sought tofurther investigate the identity and role of advanced glycosylationendproduct receptors and possible binding partners, and any consequentdiagnostic and therapeutic implications of these investigations, and itis toward this end that the present invention is directed.

SUMMARY OF THE INVENTION

In accordance with the present invention, receptor proteins aredisclosed that are derived from rat liver membranes, and which recognizeand bind to advance glycosylation endproducts. The receptor possessesthe following characteristics:

A. It recognizes and binds with the ligands AGE-RNase and AGE-CollagenI;

B. It does not recognize and bind with the ligands FFI-BSA,formaldehyde-treated BSA, glucosamide-BSA, and acetyl LDL-BSA in a solidphase ligand blotting assay; and

C. It comprises either or both of, at least two of said componentproteins, the first of said proteins having a molecular mass of about 90kD and the second of said proteins having a molecular mass of about 60kD as determined by their migration on SDS-PAGE.

The individual proteins listed above have certain commoncharacteristics, in that both proteins are also expressed on both ratmonocytes and macrophages, and both proteins copurify from elutionsbased, respectively, on an AGE ligand affinity column, an anion exchangecolumn, and a hydroxylapatite column. The proteins also have specificcharacteristics that distinguish them from each other, in that, when theproteins are immobilized on nitrocellulose in a solid phase assay suchas that disclosed herein, the 90 kD protein does not bind toAGE-modified ligands while the 60 kD protein does.

Further, the NH₂ -terminal partial amino acid sequences of each of theproteins have been prepared and confirm that each protein isdistinguishable from the other as well as from other known proteinfractions as to sequence homology. The NH₂ -terminal partial amino acidsequence for the 90 kD protein is presented below in FIG. 11 and in SEQID NO:1, and comprises a single chain of 13 amino acids including twounidentified residues. The partial amino acid sequence depicted in SEQID NO:1 is reproduced below, with X representing the unidentifiedresidues. ##STR1##

The NH₂ -terminal partial amino acid sequence for the 60 kD protein ispresented in FIG. 12 and in Sequence ID No. 2, or SEQ ID NO:2, andcomprises a single chain of 22 amino acids including two unidentifiedresidues. The partial amino acid sequence depicted in SEQ ID NO:2 isreproduced below, with X representing the unidentified residues.##STR2##

The partial DNA sequence corresponding to the partial amino acidsequences of the proteins of the present invention or a portion thereof,or a degenerate variant of such partial DNA sequence, may be prepared asa probe to screen for complementary sequences and genomic clones in thesame or alternate species, such as humans. The present invention extendsto probes so prepared that may be provided for screening cDNA andgenomic libraries for clones that may corrrespond to genes expressingthe respective proteins. For example, the probes may be prepared with avariety of known vectors. The present invention also includes thepreparation of plasmids including such vectors.

The present invention also includes full proteins having the activitiesnoted herein, and that display the partial amino acid seqences set forthand described above and with respect to SEQ ID NO:1 and SEQ ID NO:2.

In a further embodiment of the invention, the full DNA sequence of therecombinant DNA molecule or cloned gene so determined may be operativelylinked to an expression control sequence which may be introduced into anappropriate host. The invention accordingly extends to unicellular hoststransformed with the cloned gene or recombinant DNA molecule comprisinga DNA sequence encoding the 60 kD and the 90 kD protein, and moreparticularly, the complete DNA sequences determined from the partialsequences set forth above and in SEQ ID NO:1 and SEQ ID NO:2.

Likewise, the receptor proteins may be prepared alone or in operativeassociation with another molecule or pharmaceutical agent in a formsuitable for administration for either diagnostic or therapeuticpurposes. The invention therefore extends to both diagnostic andpharmaceutical compositions including the receptor and/or the proteinsdescribed herein, in combination with other diagnostic reagents in theformer instance, and in combination with pharmaceutically acceptablecarriers, and possibly, other therapeutic agents where coadministrationis deemed appropriate or desirable.

Accordingly, while the exact role that the present receptor proteinsplay in the recognition and removal of AGEs and in tissue remodeling isas yet largely undefined, its participation in the elicitation ofcertain of these activities may be strongly inferred. The receptorproteins are therefore believed to possess significant diagnostic andtherapeutic capabilities in connection with conditions involving thepresence and activity of advanced glycosylation endproducts.

The receptor proteins may be prepared by isolation and purificationtechniques from cells known to bear or produce the receptor proteins,such as rat liver cells, monocytes and peritoneal macrophage cells. Thecells or active fragments likely to participate in receptor proteinsynthesis or to have receptor proteins associated therewith may besubjected to a series of known isolation techniques, such as for exampleelution of detergent-solubilized rat liver membrane proteins from anAGE-protein affinity matrix, whereupon the present receptor componentproteins may be recovered. Naturally, alternate procedures forpreparation of the receptor proteins are contemplated and the inventionis not limited to the procedures set forth herein.

The present invention also extends to antibodies including polyclonaland monoclonal antibodies, to the receptor proteins that would find usein a variety of diagnostic and therapeutic applications. For example,the antibodies could be used to screen expression libraries to obtaingenes that encode for the receptor proteins. Further, those antibodiesthat neutralize receptor activity could initially be employed in intactanimals to better elucidate the biological role that the receptor play.Such antibodies could also participate in drug screening assays toidentify alternative drugs or other agents that may exhibit the sameactivity as the receptor proteins.

Possible therapeutic applications of the receptor proteins would includeadministration in instances where it is desirable to stimulate theremoval of advanced glycosylation endproducts and to correspondingly aidin the treatment of ailments where excess concentrations of AGEs maycause or exacerbate other dysfunctions or pathologies, such as diabetes.

The present invention also includes various diagnostic and therapeuticutilities predicated on the structure and activities of the receptorproteins. Diagnostic utilities include assays such as immunoassays withlabeled quantities of the receptor proteins, antibodies, ligands andbinding partners thereto, receptor assays, and a drug screening assay toevaluate new drugs by their ability to promote or inhibit receptorprotein production or activity, as desired. The above assays could beused to detect the presence and activity of the receptor proteins or ofinvasive stimuli, pathology or injury the presence or absence of whichwould affect receptor protein production or activity.

The present invention also extends to therapeutic methods andcorresponding pharmaceutical compositions based upon the receptorproteins, and materials having the same or an antagonistic activitythereto. Therapeutic methods would be based on the promotion of theactivities of the receptor proteins and would extend to the treatment ofdiseases or dysfunctions attributable to the absence of receptor proteinactivity, and the concomitant excess of AGEs in the host or patient suchas for example, diabetic neuropathy, renal failure, atherosclerosis,stroke, cataracts, diabetic retinopathy, and the like. This method couldbe effected with the receptor complex, its component complex, itscomponent proteins, their agonists or like drugs, or materials having apromotional effect on the production of the receptor proteins in vivo.

Therapeutic compositions comprising effective amounts of the receptorproteins, their agonists, antibodies, antagonists, or like drugs, etc.,and pharmaceutically acceptable carriers are also contemplated. Suchcompositions could be prepared for a variety of administrativeprotocols, including where appropriate, oral and parenteraladministration. Exact dosage and dosing schedule would be determined bythe skilled physician.

Diagnostic applications generally extend to a method for the measurementof protein aging both in plants and in animals, by assaying thepresence, amount, location and effect of such advanced glycosylationendproducts. Assays of plant matter and animal food samples will be ablefor example, to assess food spoilage and the degradation of otherprotein material of interest so affected, while the assays of animals,including body fluids such as blood, plasma and urine, tissue samples,and biomolecules such as DNA, that are capable of undergoing advancedglycosylation, will assist in the detection of pathology or othersystemic dysfunction.

Specifically, the methods comprise the performance of severalcompetitive assay protocols, involving the analyte, a ligand and one ormore binding partners to the advanced glycosylation endproducts ofinterest, where the binding partners are selected from the presentproteins. The binding partners may be generally selected from the groupconsisting of rat liver cells as well as monocytes and macrophage cellshaving the present receptor complex and/or the component complex and/orthe component proteins, cell components such as rat liver membranes, andthe particular cell proteins set forth herein. The cell proteins areselected from the group consisting of the 90 kD protein derived from ratliver membranes, the 60 kD protein derived from rat liver membranes, andmixtures thereof.

The ligands useful in the present invention are generally AGEderivatives that bind to AGE binding partners. These ligands may bedetected either singly and directly, or in combination with a seconddetecting partner such as avidin. Suitable ligands are selected from thereaction products of sugars such as glucose and glucose-6-phosphate withpeptides, proteins and other biochemicals such as BSA, avidin, biotin,and enzymes such as alkaline phosphatase. Other suitable ligands mayinclude synthetic AGEs or the reaction of the sugars directly withcarriers capable of undergoing advanced glycosylation. Carriers not socapable may have a synthetic AGE coupled to them. Suitable carriers maycomprise a material selected from carbohydrates, proteins, syntheticpolypeptides, lipids, bio-compatible natural and synthetic resins,antigens, and mixtures thereof.

For example, standard assays based on either cell components or the cellproteins themselves and employing extract formats may be used. Eachassay is capable of being based on enzyme linked and/or radiolabeledAGEs and their binding partners, including the AGE receptors disclosedherein. The broad format of assay protocols possible with the presentinvention extends to assays wherein no label is needed for AGEdetection. For example, one of the formats contemplates the use of abound protein-specific AGE receptor. In such instance, the analytesuspected of containing the advanced glycosylation endproducts underexamination would need only to be added to the receptor, and the boundanalyte could then be easily detected by a change in the property of thebinding partner, such as by changes in the receptor.

The assays of the invention may follow formats wherein either the ligandor the binding partner, be it a receptor or an antibody, are bound.Likewise, the assays include the use of labels which may be selectedfrom radioactive elements, enzymes and chemicals that fluoresce.

Accordingly, it is a principal object of the present invention toprovide a receptor complex for advanced glycosylation endproductsincluding receptor proteins in purified form.

It is a further object of the present invention to provide probes whichfacilitate screening of cDNA and genomic libraries in order to clone theanimal and human genes encoding the receptor proteins.

It is a still further object of the present invention to provide thecomplete nucleic acid and corresponding amino acid sequences in bothanimals and humans for the receptor complex complex and/or its componentproteins.

It is a still further object of the present invention to provideagonists, antibodies, antagonists, and analogs thereof to the receptorproteins as aforesaid, compositions including pharmaceuticalcompositions containing them and methods for their discovery andpreparation.

It is a still further object of the present invention to providepromoters of the synthesis of the receptor proteins as aforesaid, andmethods for their preparation.

It is a further object of the present invention to provide a method fordetecting the presence and amount of the receptor and/or advancedglycosylation endproducts in mammals in which invasive, spontaneous, oridiopathic pathological states related to excessive concentrations ofadvanced glycosylation endproducts are suspected to be present.

It is a further object of the present invention to provide a method andassociated assay system for screening substances such as drugs, agentsand the like, potentially effective in mimicking the activity of thereceptor.

It is a still further object of the present invention to provide amethod for the treatment of mammals to modulate the amount or activityof the receptor proteins, so as to control the consequences of suchpresence or activity.

It is a still further object of the present invention to provide amethod for the treatment of mammals to promote the amount or activity ofthe receptor proteins, so as to treat or avert the adverse consequencesof excessive concentrations of advanced glycosylation endproductsregardless of origin.

It is a still further object of the present invention to providepharmaceutical compositions for use in therapeutic methods whichcomprise or are based upon the receptor proteins or their bindingpartner(s), or upon agents or drugs that control the production and/oractivities of the receptor proteins.

It is a yet further object of the present invention to provide an assayfor the measurement of advanced glycosylation endproducts that iscapable of a broad range of alternative protocols in accordance with themethod as aforesaid.

It is a yet further object of the present invention to provide an assayas aforesaid that is capable of performance without radioactive labelsand that may be performed in automated fashion.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing description which proceedswith reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the relative binding and uptake of red bloodcells modified with various agents, and illustrating a primary aspect ofthe present invention.

FIG. 2 is a graph illustrating an assay in accordance with the presentinvention by the competitive inhibition in red blood cell binding causedby the introduction into a sample of an agent capable of stimulating redblood cells to increase their activity of recognition and removal ofadvanced glycosylation endproducts.

FIG. 3 is a bar graph illustrating the comparative uptake anddegradation of advanced glycosylation endproducts by mouse macrophagesexposed to various stimulator compounds.

FIG. 4 is a bar graph illustrating data similar to that set forth inFIG. 3, with respect to one day old human monocytes.

FIG. 5(A)--In vivo tissue distribution of ¹²⁵ I-AGE-RSA. Fifty μg ofeither ¹²⁵ I-AGE-rat serum albumin (AGE-RSA) (filled and striped bars)or ¹²⁵ I-native RSA (shaded bars) were administered i.v. to normal rats.At 10 min the animals were sacrificed, and the organs and tissues wereremoved and counted for radioactivity. Whole organ counts were correctedfor blood-associated counts as described in Methods.

FIG. 5(B)--Specific competition of ¹²⁵ I-AGE-RSA uptake in rat liver,spleen, lung and kidney. Immediately prior to receiving radio-labeledligand, rats were injected with excess non-labeled AGE-RSA (5 mg, i.v.).At the indicated time intervals, incorporated radioactivity wasdetermined in blood and tissues, and compared according to aTissue-to-Blood Isotope Ratio (TBIR) formula as described in text.Closed and striped bars: ¹²⁵ I-AGE-RSA (50 μg) alone. Open bars: ¹²⁵I-AGE-RSA (50 μg) in the presence of excess non-labeled AGE-RSA. Dataare expressed as mean±SEM of three independent measurements performed infive animals per group.

FIG. 6 depicts several graphs of an AGE-binding activity assay: FIG.6(A): Relationship of liver membrane protein concentration to AGE-ligandbinding. Aliquots (1-50 μg) of a detergent-solubilized liver membraneprotein preparation were immobilized on nitrocellulose filters,incubated with blocking buffer, and then probed for AGE-protein bindingactivity with ¹²⁵ I-AGE-BSA in the presence or absence of excessnon-labeled AGE-BSA. After washing, the blots were counted for ¹²⁵ I.Closed circles: ¹²⁵ I-AGE-BSA alone (total binding). Open circles: ¹²⁵I-AGE-BSA plus 100-fold excess non-labeled AGE-BSA (non-specificbinding). Data points represent duplicate blots. FIG. 6(B) Saturabilityof ¹²⁵ I-AGE-BSA binding. A fixed amount of solubilized liver membraneproteins (8 μg) was immobilized on duplicate nitrocellulose filterswhich were probed with increasing concentrations of ¹²⁵ I-AGE-BSA(10-100 nM, s.a.=1.5×10⁵ cpm/ng) in the presence (non-specific) orabsence (total binding) of 200-fold excess non-labeled AGE-BSA. Afterwashing, the nitrocellulose filters were counted for retained ¹²⁵ I.Specific binding was determined by subtracting non-specific binding fromtotal binding. FIG. 6(C): Scatchard analysis of specific binding data(B=pmoles/8 μg membrane protein, F=μM).

FIG. 6(D): Effects of differently modified protein ligands on ¹²⁵I-AGE-BSA binding. Filter blots of solubilized liver membranes preparedas above were probed with ¹²⁵ I-AGE-BSA (50 nM) alone, or in thepresence of 150-fold excess of various non-labeled competitors:AGE-albumin (AGE-BSA), AGE-ribonuclease (AGE-RNAse), AGE-collagen I,FFI-BSA, formaldehyde-treated albumin (f-alb), glucosamide BSA,acetyl-LDL (act-LDL). Data are expressed as the amount of ¹²⁵ I-AGE-BSA(cpm×10⁻³) retained on duplicate blots.

FIG. 7 shows the purification of rat liver AGE-binding proteins.Detergent-solubilized membrane proteins were fractionated by successivePEI-cellulose, DEAE-cellulose, and BSA-Sepharose 4B columnchromatography, as described in Methods. The flow-through from theBSA-Sepharose column was then applied to an AGE-BSA-Sepharose 4B column.This column was washed and bound proteins were eluted by the addition ofhigh salt buffer. Each fraction was concentrated and analyzed forAGE-binding activity using the binding assay described in FIG. 6. Opencircles: total binding activity; closed circles: non-specific activity.Inset: SDS-PAGE analysis of fraction #8 (mercaptoethanol reduced), andstained with Coommassie Blue.

FIG. 8--(A) Ligand blot analysis of rat liver membrane proteins.Aliquots (15 μg each) of detergent-solubilized membrane proteins wereelectrophoresed through 8-16% acrylamide gradient gel under non-reducingconditions and eletro-transferred onto nitrocellulose filters. Using ¹²⁵I-AGE-BSA (50 nM, 8.0×10⁵ cpm/ng) as probe, specific binding wasdetermined in the presence of 0, 25, or 150-fold excess non-labeledAGE-BSA using autoradiographic detection. Migration of molecular weightstandards is shown at left. FIG. 8(B) Aliquots of solubilized membraneproteins (mercaptoethanol reduced), were electrophoresed through an8-16% gradient gel and electro-transferred onto nitrocellulose filters.After washing and blocking with excess BSA, the blots were probed withpurified IgG fractions of either anti-p60 or anti-p90 chicken antiseraor the corresponding preimmune sera, and then exposed to goatanti-chicken antibody conjugated to alkaline phosphatase and reacted forphosphatase-dependent color development. Lane 1, preimmune IgG; Lane 2,anti-p60 IgG; Lane 3, preimmune IgG, Lane 4, anti-p90 IgG. Results arerepresentative of three independent experiments.

FIG. 9--Demonstration by flow cytometry of expression of p60 and p90AGE-binding proteins rat monocytes and macrophages. Peripheral bloodmonocytes (A-D) and peritoneal resident macrophages (E and F) weretreated with biotinylated anti-p60 alone (A and E), biotinylatedanti-p90 alone (B and F), biotinylated anti-p60+20-fold excessunconjugated anti-p90 (C), biotinylated anti-p90+20-fold excessunconjugated anti-p60 (D), followed by FITC-avidin and analyzed byFACSCAN. Arrow 1: Fluorescence of cells treated with FITC-avidin in theabsence of either anti-p60 or anti-p90 (dotted line). Arrow 2:Fluorescence of cells treated with FITC-avidin subsequent to treatmentwith biotinylated chicken IgG (isotypic control). Arrow 3: Fluorescenceof cells treated with FITC-avidin subsequent to treatment of cells withbiotinylated anti-p60 (A,C,E) and anti-p90 (B,D,F). Note the lack ofcompetition between a 20-fold excess of unconjugated anti-p90 for thebinding of anti-p60 to the monocytes (panel A vs C), and the lack ofcompetition between a 20-fold excess of unconjugated anti-p60 for thebinding of anti-p90 to the monocyte cell surface (panel B vs D). Allantibodies were used at a final concentration of 5 μg/ml, unlessotherwise indicated.

FIG. 10--Inhibition of ¹²⁵ I-AGE-BSA binding (FIG. 10A) and ¹²⁵I-FFI-BSA binding (FIG. 10B) on rat macrophage cell surface by anti-p60and anti-p90 antibodies. Rat peritoneal resident macrophages werecollected by peritoneal lavage and purified, then incubated with theindicated radio-labeled ligand in the presence or absence of a 10-foldexcess of non-labeled ligand or in the presence of antibodies p60 orp90-specific at the indicated dilutions. Both antibodies were used alone(undiluted: 2 μg/200 μl) or in combination (at 1:10 dilution). Data areexpressed as % of maximal control binding (defined as the amount of ¹²⁵I-ligand bound to the cell surface in the presence of 10% FBS) andrepresent the mean of duplicate experiments.

FIG. 11 depicts the NH₂ -terminal partial amino acid sequence preparedby blotting a quantity of gel-purified rat liver membrane protein havinga molecular mass of about 90 kD (p90) onto Immobilon membranes. Theamino acids are numbered from 1 to 13. This sequence is identicallydepicted in the SEQUENCE LISTING presented later on herein, inaccordance with 37 C.F.R. 1.821-825, enacted Oct. 1, 1990, and iscumulatively and alternately referred to as SEQ ID NO:1.

FIG. 12 depicts the NH₂ -terminal partial amino acid sequence preparedby blotting a quantity of gel-purified rat liver membrane protein havinga molecular mass of about 60 kD (p60) onto Immobilon membranes. Theamino acids are numbered from 1 to 22. This sequence is identicallydepicted in the SEQUENCE LISTING presented later on herein, inaccordance with 37 C.F.R. 1.821-825, enacted Oct. 1, 1990, and iscumulatively and alternately referred to as SEQ ID NO:2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,"Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: APractical Approach," Volumes I and II (D. N. Glover ed. 1985);"Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic AcidHybridization" (B. D. Hames & S. J. Higgins eds. 1985); "TranscriptionAnd Translation" (B. D. Hames & S. J. Higgins eds. 1984); "Animal CellCulture" (R. I. Freshney ed. 1986); "Immobilized Cells And Enzymes" (IRLPress, 1986); B. Perbal, "A Practical Guide To Molecular Cloning"(1984).

Therefore if appearing herein, the following terms shall have thedefinitions set out below.

The amino acid residues described herein are preferred to be in the "L"isomeric form. However, residues in the "D" isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfuctional property of immunoglobulin-binding is retained by thepolypeptide. NH2 refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

    ______________________________________                                        TABLE OF CORRESPONDENCE                                                       SYMBOL                                                                        1-Letter  3-Letter        AMINO ACID                                          ______________________________________                                        Y         Tyr             tyrosine                                            G         Gly             glycine                                             F         Phe             phenylalanine                                       M         Met             methionine                                          A         Ala             alanine                                             S         Ser             serine                                              I         Ile             isoleucine                                          L         Leu             leucine                                             T         Thr             threonine                                           V         Val             valine                                              P         Pro             proline                                             K         Lys             lysine                                              H         His             histidine                                           Q         Gln             glutamine                                           E         Glu             glutamic acid                                       W         Trp             tryptophan                                          R         Arg             arginine                                            D         Asp             aspartic acid                                       N         Asn             asparagine                                          C         Cys             cysteine                                            ______________________________________                                    

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

As used throughout the present application, the terms "receptor complex"and "receptor complex/component protein(s)" includes both the singularand plural and contemplates the existence of a single receptor structurecomprised as all or part thereof, of the individual proteins definedherein, or a plurality of receptor structures respectively constitutedin whole or in part by the individual proteins. This definition istherefore to be explicitly distinguished from the definition that may beinferred from the term as it appears in the manuscript by Yang et al.from which the present application is drawn in part.

An "antibody" is any immunoglobulin, including antibodies and fragmentsthereof, such as Fab, F(ab')₂ or dAb, that binds a specific epitope. Theterm encompasses, inter alia, polyclonal, monoclonal, and chimericantibodies, the last mentioned described in further detail in U.S. Pat.Nos. 4,816,397 and 4,816,567. An antibody preparation is reactive for aparticular antigen when at least a portion of the individualimmunoglobulin molecules in the preparation recognize (i.e., bind to)the antigen. An antibody preparation is non-reactive for an antigen whenbinding of the individual immunoglobulin molecules in the preparation tothe antigen is not detectable by commonly used methods.

In its broadest aspect, the present invention relates to receptorcomplex proteins as defined herein that are derived from rat livermembranes, as well as monocytes and peritoneal macrophage cells, andthat recognize and bind advanced glycosylation endproducts. The receptorhas the following characteristics:

A. It recognizes and binds with the ligands AGE-RNase and AGE-CollagenI;

B. It does not recognize and bind with the ligands FFI-BSA,formaldehyde-treated BSA, glucosamide-BSA, and acetyl LDL-BSA in a solidphase ligand blotting assay; and

C. It comprises either or both of at least two proteins (the conponentproteins), the first of said proteins having a molecular mass of about90 kD and the second of said proteins having a molecular mass of about60 kD as determined by their migration on SDS-PAGE.

The receptor proteins have certain common characteristics, in that bothproteins are also expressed on both rat monocytes and macrophages, andboth proteins copurify from elutions based, respectively, on an AGEligand affinity column, an anion exchange column, and a hydroxylapatitecolumn. The proteins also have specific characteristics that distinguishthem from each other, in that, when the proteins are immobilized onnitrocellulose in a solid phase assay, the 90 kD protein does not bindto AGE-modified ligands while the 60 kD protein does.

As set forth earlier, the NH₂ -terminal partial amino acid sequences ofeach of the component proteins have been prepared and confirm that eachprotein bears no homology with the other as well as with other knownprotein fractions. The NH₂ -terminal partial amino acid sequence for the90 kD protein is presented below and in SEQ ID NO:1, and comprises asingle chain of 13 amino acids including two unidentified residuesrepresented by "X". ##STR3##

The NH₂ -terminal partial amino acid sequence for the 60 kD protein ispresented below in SEQ ID NO:2, and comprises a single chain of 22 aminoacids including two unidentified residues, likewise represented by "X".##STR4##

As stated earlier, the partial DNA sequence corresponding to the partialamino acid sequences of the proteins of the present invention or aportion thereof, may be prepared as a probe to screen for complementarysequences and genomic clones in the same or alternate species, such ashumans. The present invention extends to probes so prepared that may beprovided for screening cDNA and genomic libraries for clones that maycorrrespond to genes expressing the respective proteins. For example,the probes may be prepared with a variety of known vectors, such asphage I vectors. The present invention also includes the preparation ofplasmids including such vectors.

The present invention also includes full proteins having the activitiesnoted herein, and that display the partial amino acid seqences set forthand described above and with respect to SEQ ID NO:1 and SEQ ID NO:2.

As stated earlier, the receptor proteins may be prepared by isolationand purification from cells known to bear or produce the proteins, suchas rat liver cells, monocytes and peritoneal macrophage cells. The cellsor active fragments likely to participate in receptor complex/proteinsynthesis or to have receptor proteins associated therewith may besubjected to a series of known isolation techniques, such as for exampleelution of detergent-solubilized rat liver membrane proteins from anAGE-protein affinity matrix, whereupon the proteins may be recovered. Aspecific protocol is set forth by way of illustration in Example III,later on herein. The present invention naturally contemplates alternatemeans for preparation of the proteins, including stimulation of complexproducer cells with promoters of receptor protein synthesis followed bythe isolation and recovery of the receptor proteins as indicated above,as well as chemical synthesis, and the invention is accordingly intendedto cover such alternate preparations within its scope.

The present invention also extends to antibodies including polyclonaland monoclonal antibodies, to the receptor proteins that would find usein a variety of diagnostic and therapeutic applications. For example,the antibodies could be used to screen expression libraries to obtainthe gene that encodes either the receptor complex or proteins. Further,those antibodies that neutralize receptor protein activity couldinitially be employed in intact animals to better elucidate thebiological role that the receptor proteins play. Such antibodies couldalso participate in drug screening assays to identify alternative drugsor other agents that may exhibit the same activity as the receptorproteins.

Both polyclonal and monoclonal antibodies to the receptor complex arecontemplated, the latter capable of preparation by well known techniquessuch as the hybridoma technique, utilizing, for example, fused mousespleen lymphocytes and myeloma cells. Immortal, antibody-producing celllines can also be created by techniques other than fusion, such asdirect transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus. Specific avian polyclonalantibodies were raised herein and are set forth in Example III.Naturally, these antibodies are merely illustrative of antibodypreparations that may be made in accordance with the present invention.

As the receptor proteins appear to play a role in the recognition andremoval of advanced glycosylation endproducts in vivo, the presentinvention contemplates both diagnostic and therapeutic applications forthese agents. Accordingly, the receptor proteins may be prepared for usein a variety of diagnostic methods, set forth in detail hereinafter, andmay be labeled or unlabeled as appropriate. Likewise, the receptorproteins may be prepared for administration in various scenarios fortherapeutic purposes, in most instances to assist in reducing theconcentration of AGEs in vivo.

The receptor proteins may be prepared in a therapeutically effectiveconcentration as a pharmaceutical composition with a pharmaceuticallyacceptable carrier. Other compatible pharmaceutical agents may possiblybe included, so that for example certain agents may be simultaneouslycoadministered. Also, the receptor proteins may be associated with orexpressed by a compatible cellular colony, and the resulting cellularmass may then be treated as a therapeutic agent and administered to apatient in accordance with a predetermined protocol. Numeroustherapeutic formulations are possible and the present inventioncontemplates all such variations within its scope. A variety ofadministrative techniques may be utilized, among them topicalapplications as in ointments or on surgical and other topical appliancessuch as, surgical sponges, bandages, gauze pads, and the like. Also,such compositions may be administered by parenteral techniques such assubcutaneous, intravenous and intraperitoneal injections,catheterizations and the like.

Corresponding therapeutic utilities take advantage of the demonstratedactivity of the proteins toward advanced glycosylation endproducts.Thus, to the extent that the in vivo recognition and removal of AGEsserves to treat ailments attributable to their presence in an excessconcentration, the administration of the present receptor proteinscomprises an effective therapeutic method. Such conditions as diabeticnephropathy, renal failure and the like may be treated and/or averted bythe practice of the therapeutic methods of the present invention.Average quantities of the active agent may vary and in particular shouldbe based upon the recommendations and prescription of a qualifiedphysician or veterinarian, with an exemplary dosage regimen extending toup to about 25 mg/kg/day.

The present invention also relates to a variety of diagnosticapplications, including methods for the measurement of the presence andamount of advanced glycosylation endproducts in both plants and animals,including humans. The methods comprise assays involving in addition tothe analyte, one or more binding partners of the advanced glycosylationendproducts, and one or more ligands.

Accordingly, the present assay method broadly comprises the steps of:

A. preparing at least one biological sample suspected of containing saidadvanced glycosylation endproducts;

B. preparing at least one corresponding binding partner directed to saidsamples, wherein said binding partner includes or is selected from thepresent receptor proteins, and mixtures;

C. placing a detectible label on a material selected from the groupconsisting of said samples, a ligand to said binding partner and saidbinding partner;

D. placing the labeled material from Step C in contact with a materialselected from the group consisting of the material from Step C that isnot labeled; and

E. examining the resulting sample of Step D for the extent of binding ofsaid labeled material to said unlabeled material.

Suitable analytes may be selected from plant matter, blood, plasma,urine, cerebrospinal fluid, lymphatic fluid, and tissue; and thecompounds FFI and AFGP, individually and bound to carrier proteins suchas the protein albumin. The analyte may also comprise a syntheticallyderived advanced glycosylation endproduct which is prepared, forexample, by the reaction of a protein or other macromolecule with asugar such as glucose, glucose-6-phosphate, or others. This reactionproduct could be used alone or could be combined with a carrier in thesame fashion as the FFI-albumin complex.

The carrier may be selected from the group consisting of carbohydrates,proteins, synthetic polypeptides, lipids, bio-compatible natural andsynthetic resins, antigens and mixtures thereof.

As stated earlier, the present invention seeks by means of the presentreceptor proteins to diagnose both the degradative effects of advancedglycosylation of proteins in plants and the like, and the adverseeffects of the buildup of advanced glycosylation endproducts in animals.Such conditions as age- or diabetes-related hardening of the arteries,skin wrinkling, arterial blockage, and diabetic, retinal and renaldamage in animals all result from the excessive buildup or trapping thatoccurs as advanced glycosylation endproducts increase in quantity.Therefore, the diagnostic method of the present invention seeks to avertpathologies caused at least in part by the accumulation of advancedglycosylation endproducts in the body by monitoring the amount andlocation of such AGEs.

Likewise, as advanced glycosylation endproducts may be measured by theextent that they bind to receptors on cells from a variety of sources,the assays of the present invention are particularly suited to designand performance around this activity. For example, in a typicalcompetitive assay in accordance with the present invention, the presentreceptor and/or cellular material bearing the receptor may be combinedwith the analyte and the ligand and the binding activity of either orboth the ligand or the analyte to the receptor may then be measured todetermine the extent and presence of the advanced glycosylationendproduct of interest. In this way, the differences in affinity betweenthe components of the assay serves to identify the presence and amountof the AGE.

The present invention also relates to a method for detecting thepresence of stimulated, spontaneous, or idiopathic pathological statesin mammals, by measuring the corresponding presence of advancedglycosylation endproducts. More particularly, the activity of AGEs maybe followed directly by assay techniques such as those discussed herein,through the use of an appropriately labeled quantity of at least one ofthe binding partners to AGEs as set forth herein. Alternately, AGEs canbe used to raise binding partners or antagonists that could in turn, belabeled and introduced into a medium to test for the presence and amountof AGEs therein, and to thereby assess the state of the host from whichthe medium was drawn.

Thus, both AGE receptors and any binding partners thereto that may beprepared, are capable of use in connection with various diagnostictechniques, including immunoassays, such as a radioimmunoassay, usingfor example, a receptor or other ligand to an AGE that may either beunlabeled or if labeled, then by either radioactive addition, reductionwith sodium borohydride, or radioiodination.

In an immunoassay, a control quantity of a binding partner to advancedglycosylation endproducts may be prepared and optionally labeled, suchas with an enzyme, a compound that fluoresces and/or a radioactiveelement, and may then be introduced into a tissue or fluid sample of amammal believed to be undergoing invasion. After the labeled material orits binding partner(s) has had an opportunity to react with sites withinthe sample, the resulting mass may be examined by known techniques,which may vary with the nature of the label attached.

The presence of AGE activity in animals and plants can be ascertained ingeneral by immunological procedures are which utilize either a bindingpartner to the advanced glycosylation endproduct or a ligand thereto,optionally labeled with a detectable label, and further optionallyincluding an antibody Ab₁ labeled with a detectable label, an antibodyAb₂ labeled with a detectable label, or a chemical conjugate with abinding partner to the advanced glycosylation endproduct labeled with adetectable label. The procedures may be summarized by the followingequations wherein the asterisk indicates that the particle is labeled,and "BP" in this instance stands for all binding partners of advancedglycosylation endproduct(s) under examination:

A. BP*+Ab₁ =BP*Ab₁

B. BP+Ab*=BPAb₁ *

C. BP+Ab₁ +Ab₂ *=BPAb₁ Ab₂ *

D. Carrier*BP+Ab₁ =Carrier*BPAb₁

These general procedures and their application are all familiar to thoseskilled in the art and are presented herein as illustrative and notrestrictive of procedures that may be utilized within the scope of thepresent invention. The "competitive" procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Optional procedureC, the "sandwich" procedure, is described in U.S. Pat. Nos. RE 31,006and 4,016,043, while optional procedure D is known as the "doubleantibody", or "DASP" procedure.

A further alternate diagnostic procedure employs multiple labeledcompounds in a single solution for simultaneous radioimmune assay. Inthis procedure disclosed in U.S. Pat. No. 4,762,028 to Olson, acomposition may be prepared with two or more analytes in a coordinatedcompound having the formula: radioisotope-chelator-analyte.

In each instance, the advanced glycoslation endproduct forms complexeswith one or more binding partners and one member of the complex may belabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by the knownapplicable detection methods.

With reference to the use of an AGE antibody as a binding partner, itwill be seen from the above that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. Where used and for purposes of this description, Ab₁will be referred to as a primary or anti-advanced glycosylationendproduct antibody, and Ab₂ will be referred to as a secondary oranti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

Suitable radioactive elements may be selected from the group consistingof ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵I, ¹³¹ I, and ¹⁸⁶ Re. In the instance where a radioactive label, such isprepared with one of the above isotopes is used, known currentlyavailable counting procedures may be utilized.

In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectro-photometric, thermometric,amperometric or gasometric techniques known in the art. The enzyme maybe conjugated to the advanced glycosylation endproducts, their bindingpartners or carrier molecules by reaction with bridging molecules suchas carbodiimides, diisocyanates, glutaraldehyde and the like. Also, andin a particular embodiment of the present invention, the enzymesthemselves may be modified into advanced glycosylation endproducts byreaction with sugars as set forth herein.

Many enzymes which can be used in these procedures are known and can beutilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase, hexokinase plus GPDase, RNAse, glucose oxidase plus alkalinephosphatase, NAD oxidoreductase plus luciferase, phosphofructokinaseplus phosphoenol pyruvate carboxylase, aspartate aminotransferase plusphosphoenol pyruvate decarboxylase, and alkaline phosphatase. U.S. Pat.Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way ofexample for their disclosure of alternative labeling material andmethods.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine and auramine.A particular detecting material is anti-rabbit antibody prepared ingoats and conjugated with fluorescein through an isothiocyanate.

The present invention includes assay systems that may be prepared in theform of test kits for the quantitative analysis of the extent of thepresence of advanced glycosylation endproducts. The system or test kitmay comprise a labeled component prepared by one of the radioactiveand/or enzymatic techniques discussed herein, coupling a label to abinding partner to the advanced glycosylation endproduct such as areceptor or ligand as listed herein; and one or more additionalimmunochemical reagents, at least one of which is a free or immobilizedligand, capable either of binding with the labeled component, itsbinding partner, one of the components to be determined or their bindingpartner(s).

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of advanced glycosylation endproducts. In accordancewith the testing techniques discussed above, one class of such kits willcontain at least labeled AGE, or its binding partner as stated above,and directions, of course, depending upon the method selected, e.g.,"competitive", "sandwich", "DASP" and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

For example, a first assay format contemplates a bound receptor to whichare added the ligand and the analyte. The resulting substrate is thenwashed after which detection proceeds by the measurement of the amountof ligand bound to the receptor. A second format employs a bound ligandto which the receptor and the analyte are added. Both of the first twoformats are based on a competitive reaction with the analyte, while athird format comprises a direct binding reaction between the analyte anda bound receptor. In this format a bound receptor-specific carrier orsubstrate is used. The analyte is first added after which the receptoris added, the substrate washed, and the amount of receptor bound to thesubstrate is measured.

More particularly, the present invention includes the following protocolwithin its scope:

A method for determining the amount of advanced glycosylationendproducts in an analyte comprising:

A. providing a sample of monocytes bearing the present receptorcomplex/component protein(s);

B. inoculating said sample with a known advanced glycosylationendproduct bound to a whole cell; and

C. counting the whole cells of Step B that are bound to and/orinternalized by said sample.

The specific protocol set forth above is illustrated in the examplesthat follow later on herein, and reflects the broad latitude of thepresent invention. All of the protocols disclosed herein may be appliedto the qualitative and quantitative determination of advancedglycosylation endproducts and to the concomitant diagnosis andsurveillance of pathologies in which the accretion of advancedglycosylation endproducts is implicated. Such conditions as diabetes andthe conditions associated with aging, such as atherosclerosis and skinwrinkling represent non-limiting examples, and accordingly methods fordiagnosing and monitoring these conditions are included within the scopeof the present invention.

Accordingly, a test kit may be prepared for the demonstration of thepresence and activity of AGEs, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of anadvanced glycoslation endproduct binding partner comprising the presentreceptor protein(s) or a specific binding partner thereto, to adetectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of a binding partner as described above, or a ligandthereof, generally bound to a solid phase to form an immunosorbent, orin the alternative, bound to a suitable tag, or plural such endproducts, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may comprise:

(a) a labeled component which has been obtained by coupling the abovebinding partner to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeledcomponent (a);

(iii) a ligand capable of binding with at least one of the component(s)to be determined; and

(iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the advanced glycosylation endproduct and the bindingpartner.

The present invention will be better understood from a consideration ofthe following illustrative examples and data. Accordingly, Examples Iand II presented in parent application Ser. No. 453,958 confirm thebasic hypothesis that the in vivo recognition and removal of AGEs isreceptor mediated, and Examples III presents the investigations andexperiments that have resulted in the identification of theliver-derived AGE receptors of the present invention.

EXAMPLE I

In this example, the existence of the receptor-mediated clearance systemof advanced glycosylation endproducts that underlies the present assaywas initially explored, in part, by the performance of a competitivephagocytosis assay was conducted with whole monocytes. A full review ofthe details of the experimental procedures involved is presented in U.S.Pat. No. 4,900,747, and reference may be made thereto for such purpose.

In this example, human red blood cells (RBCs) were collected andisolated, and separate quantities were prepared to facilitate theperformance of the assay. Specifically, a quantity of RBCs wereopsonized by incubation with an appropriate antiserum. A furtherquantity was bound to the advanced glycosylation endproduct FFI by acarbodiimide bond, and additional RBCs were separately glycosylated byreactions with glucose, glucose-6-phosphate, xylose and arabinose,respectively. Lastly, AGE-BSA and human monocytes were prepared.Phagocytosis assays proceeded by the incubation of the RBCs with themonocyte cultures followed by fixation of the sample wells and lastlycounting under 40× phase microscopy.

An FFI-RBC half life assay was also conducted with Balb/c mice that wereinoculated with FFI-RBC suspensions labeled with ⁵¹ Cr. The labeledcells were washed at least four times to remove unbound isotope. TwelveBalb/c mice were then injected intravenously with 200 μl RBC suspension.Each sample was administered in three Balb/c mice. At appropriate timeintervals the mice were bled (0.2 ml) and radioactivity levels weremeasured by counting.

RESULTS

Maximum binding of red cells was observed on Day-7 of monocyteincubation in vitro. Maximum binding and endocytosis of FFI-RBC wascomplete within 30-45 minutes while opsonized cells were maximally boundwithin 15 minutes. At the end of one hour incubation of FFI-coupledRBC's with cultured human monocytes, per cent phagocytosis andphagocytic index were estimated. As shown in FIG. 1, %erythrophagocytosis of FFI-modified red cells (55%) and IgG-coated redcells (70%) were significantly higher than that of control PBS-treatedcells (4%). Similarly the phagocytic index of FFI-treated RBC's wasgreatly elevated (3.4) as compared to normal controls (1.2).

In order to establish the specificity of the interaction of FFI-RBC'swith the human monocytes, competition experiments were carried out inwhich binding and ingestion of red cells was observed in the absence andpresence of AGE-BSA, prepared as described in Methods (Vlassara et al.,supra.). As shown in FIG. 2, the addition of AGE-BSA at concentrationsof 500 mg/ml inhibited the FFI-RBC binding by more than 70% of thecontrol. In contrast, AGE-BSA did not inhibit opsonized or PBS-treatedred cells, even at maximal concentrations (1 mg/ml). These datasuggested that FFI-modified red cells were recognized and boundspecifically by the monocyte AGE-binding site, and consequentlyconfirmed the operability of the present assay.

DISCUSSION

The above tests extend previous observations on the recognition ofadvanced glycosylation endproducts (AGE) by a specificmonocyte/macrophage receptor, and present evidence that such adductsonce attached chemically or formed in vitro on the surface of intacthuman cells can induce cell binding and ingestion by normal humanmonocytes. The experiments establish the development of a competitivereceptor-based assay for AGEs measuring by way of illustration herein,AGE-red cell binding in the presence of large excess of AGE-BSA(Vlassara et al., supra.).

EXAMPLE II

This example comprises a series of experiments that were initiallyperformed to measure the ability of agents to stimulate phagocytic cellsto stimulate uptake and degradation of endproducts (AGEs), and therebyfurther confirmed the hypothesis that this activity isreceptor-mediated.

Several AGEs were prepared using the same procedure as disclosed inExample I, above. Accordingly, FFI-HA was prepared as described andquantities were bound to both human and bovine albumin. A water solublecarbodiimide was used to attach the acid moiety of the FFI-HA to anamino group on the protein. After preparation, the conjugate waspurified and then used in vitro to stimulate macrophages, by incubationfor from 4 to 24 hours.

The AGEs that were to be observed for uptake and degradation wereappropriately radiolabeled so that they could be traced. Thereafter, thestimulated macrophages were tested by exposure to the radiolabeled AGEsfollowing exposure to various agents to measure the effect that theseagents had on the ability of the macrophages to take up and degrade thelabeled AGEs. The above procedures conform to the protocol employed byVlassara et al., supra, and confirmed that a competitive assay based ona cellular receptor for AGEs is feasible.

It was also demonstrated that monocyte or macrophage cells can also bestimulated by AGE-carrier molecules which result in cells with enhancedability to bind, internalize and degrade other AGE-molecules.AGE-carrier molecules are made, for example, from the reaction ofglucose or glucose-6-phosphate with albumin. After purification of thereaction product, the AGE-albumin uptake of AGE-macromoleculesdemonstrated as in (A) above. AGE-BSA (prepared from the incubation ofglucose-6-phosphate with albumin for 6-8 weeks) at 0.1 mg/ml demostratesa stimulatory effect on AGE-BSA uptake by human monocytes (FIG. 4, barAA), and shows a slight stimulation at higher concentrations (bars BBand CC). This observation further supports the role of these ligands inconjunction with cellular receptors and point to the application ofthese agents in a competitive AGE assay protocol.

EXAMPLE III

The following example discloses the purification from rat liver, andpartial amino acid sequencing, of two membrane proteins of approximately60 and 90 kD, respectively, that bind AGE-modified proteins. Both of theproteins presented are expressed on the surface of rat monocytes andresident peritoneal macrophages, suggesting a relationship to theAGE-receptor system earlier identified on these cells. The proteins arebelieved to be involved in tissue repair and remodelling.

MATERIALS AND METHODS

Chemicals and reagents. Bovine serum albumin (BSA) (Fraction V), bovineribonuclease, glucosamide-BSA, glucose-6-phosphate and collagen I werepurchased from Sigma Chemical Co. (St. Louis, Mo.). Triton X-114 andTriton X-100 were purchased from Boehringer Mannheim Biochemicals(Indianapolis, Ind.). Sodium ¹²⁵ iodide was obtained from New EnglandNuclear (Boston, Mass.). Nitrocellulose membranes were from Schliecher &Schuell (Keene, N.H.). CNBr-activated Sepharose 4B was purchased fromPierce (Rockford, Ill.). LDL and acetyl-LDL were the generous gift ofDr. David Via (Baylor College of Medicine, Houston, Tex.).2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) and its derivative,FFI-1-hexanoic acid (FFI-HA) were kindly provided by Dr. Peter Ulrich(Geritech Inc., Northvale, N.J.).

Preparation of AGE-proteins and formaldehyde-modified proteins. AGE-BSA,AGE-RSA, AGE-ribonuclease and AGE-collagen I were generated bystandardized methods as previously described in detail. In brief, eachprotein solution (25 mg/ml) was incubated with either 0.5M glucose orglucose-6-phosphate in 100 mM phosphate buffer (pH 7.4) at 37° C. forsix weeks under sterile conditions, and then low molecular weightreactants were removed by dialysis against phosphate buffered saline(PBS; 20 mM sodium phosphate buffer containing 0.15M NaCl, pH 7.4).FFI-HA was coupled to BSA with the water-soluble carbodiimide, EDAC, asdescribed previously. Characteristic fluorescence of the AGE-proteinswas observed at 450 nm upon excitation at 390 nm. Formaldehyde-modifiedBSA was prepared as described, by incubating BSA in 0.1M sodiumcarbonate buffer (pH 10) with 0.33M formaldehyde at 37° C. for 5 hours,followed by extensive dialysis against PBS. All protein concentrationswere determined by the method of Bradford. Protein and AGE-proteinpreparations were radio-labeled with ¹²⁵ I! by the Iodo-Gen method forexample, 2 mg of AGE-BSA were incubated with 25 mCi carrier-free ¹²⁵ I!in an Iodogen-coated glass vial at room temperature for 45 min. In orderto separate free from bound ¹²⁵ I!, the sample was fractionated bySephadex G-25M column chromatography and dialysis against PBS, until atleast 98% of the ¹²⁵ I was trichloroacetic acid-precipitable. Thespecific radioactivity of the labeled ¹²⁵ I-AGE-BSA was between8,000-15,000 cpm/ng protein.

BSA and AGE-BSA Sepharose were prepared by reacting either BSA orAGE-BSA (25 mg/ml) with CNBr-Sepharose gel (5 ml/g of dry powder)according to the manufacturers instructions, in coupling buffer (0.1MNaHCO₃, 0.5M NaCl, pH 8.3). The mixture was rotated for 2 h at roomtemperature. Excess ligand was washed from the resin with couplingbuffer and then with Tris-HCl buffer (0.1M, pH 8.0) for 2 h at roomtemperature to block remaining active groups. The resin was then washedwith three cycles of sodium acetate buffer (0.1M, pH 4) containing NaCl(0.5M), followed by Tris-HCl buffer (0.1M, pH 8) containing NaCl (0.5M),and stored at 4°-8° C.

In Vivo Studies. For tissue distribution studies, AGE-rat serum albumin(AGE-RSA) was prepared as described above, and radioiodinated to aspecific activity of 8.3×10⁵ cpm/μg. Similarly, normal RSA was iodinatedto a specific activity of 6.2×10⁵ cpm/μg. Freshly drawn rat red bloodcells (RBC) were labeled with ⁵¹ Cr to allow subsequent correction fortissue counts for blood-associated radioactivity. Approximately 1 mCi of⁵¹ Cr was added to 10 ml RBC, mixed thoroughly and allowed to incubatefor 30 min at room temperature prior to washing with PBS containing 1%BSA and 0.1% dextrose until supernatant radioactivity was less than 1%of that in the packed RBC. Buffer was added to labeled RBC to obtain ahematocrit of 40% and the labeled RBC were used immediately.

To determine the tissue distribution of AGE-ligands, normal maleSprague-Dawley rats (200 g) were divided into groups of five, andanesthetized with sodium pentobarbital (40 mg/kg). All animals receivedan injection of ⁵¹ Cr-RBC (0.65 ml, i.v.) five minutes prior to thelabeled ligand. The indicated groups of rats received either ¹²⁵I-AGE-RSA (50 μg in 0.1 ml, i.v.) or an identical amount of ¹²⁵ I-normalRSA. At the indicated time intervals, 0.5 ml aliquots of blood weredrawn and various organs were removed and counted for radioactivity. Thespecificity of AGE-ligand uptake in various organs was assessed byinjecting groups of animals with excess non-labeled AGE-BSA (5 mg) twominutes prior to administration of 50 μg of ¹²⁵ I-AGE-BSA. After 5, 20,and 60 minutes, 0.5 ml blood samples were collected, animals weresacrificed, and various organs and tissues were collected and countedfor radioactivity. The red blood cells were lysed with water, andprotein was precipitated with 20% TCA. The organs were weighed,homogenized with a hand homogenizer, protein was precipitated with 20%TCA and counted for radioactivity. The tissue-to-blood isotope ratio(TBIR) was calculated by the formula TBIR= ¹²⁵ I/⁵¹ Cr in tissue!/ ¹²⁵I/⁵¹ Cr in blood!. TBIR is a dimensionless index of the degree to whichany tissue sample has sequestered labeled ligand relative to the blood.

Solubilization and fractionation of hepatic membrane proteins. Livermembranes were prepared according to the method of Thom et al. with somemodifications. For a typical membrane preparation, 14 grams of rat liverwere homogenized in 80 ml TNE buffer 50 mM Tris-HCl buffer (pH 8.0),containing 150 mM NaCl, 0.1 mM EDTA, and 23 μg/ml phenylmethylsulfonylfluoride (PMSF)! and centrifuged for 10 min at 3000×g. The supernate waslayered on top of a solution of 40% sucrose in TNE buffer, andcentrifuged at 24,000×g for 1 h at 4° C. The membranes were collectedfrom the interface with a Pasteur pipette. The membrane preparation wassolubilized in TNE buffer containing 2% Triton X-114 at 4° C. andclarified by centrifugation for 30 min at 100,000×g. The supernate wasthen warmed to 30° C. and the detergent phase, aqueous phase anddetergent-insoluble pellet were separated according to the phaseseparation method described by Bordier. The resulting detergent phasewas either used directly for purification of AGE-binding proteins ordiluted 1:10 with PBS containing 2% Triton X-100 and 2 mM PMSF. Thismaterial (D-phase) was frozen at -80° C. until further use.

In Vitro solid-phase AGE binding assay. AGE-binding activity wasdetermined by a modified version of solid-phase binding assay developedfor the IL-1 receptor. Aliquots of detergent-solubilized membraneproteins were blotted onto grid-marked nitrocellulose (NC) membranes.The blots were dried at room temperature and could be stored at roomtemperature for several weeks without apparent loss of binding activity.The NC membranes were cut into small squares (0.9 cm²) with theimmobilized protein at the center and distributed in 24-well trays(Costar, Cambridge, Mass.). Immobilized protein was reconstituted inPBS, pH 7.4, containing 0.5% Triton X-100 for 30-60 min at roomtemperature. The blots were subsequently incubated in blocking buffer(PBS, pH 7.4, containing 2% BSA, 0.2% Triton X-100 and 1 mM MgCl₂) for 2h with agitation at 4° C. Specific ligand binding was carried out byadding 50-100 nM ¹²⁵ I-AGE-BSA directly to the blocking buffer andagitating at 4° C for a further 1.5 h. The NC membranes were transferredto a new tray and rinsed quickly 3 times with PBS containing 0.2% TritonX-100, followed by two additional 10 min. washings with PBS. Ligandbinding was then evaluated by autoradiography or gamma counting.

Ligand blotting. SDS-PAGE and electro-blotting were performed aspreviously described. Proteins were electrophoretically separated oneither 8-16% or 4-20% gradient polyacrylamide gels. Afterelectro-blotting the proteins from the gel onto NC membranes, the blotswere washed at 4° C. overnight with PBS containing 0.2% Triton X-100.The blots were then incubated in blocking buffer for 3 h at 4° C. withagitation. Ligand binding was performed by adding 10 nM ¹²⁵ 1-AGE-BSA tothe blocking buffer. After 1.5-2 h at 4° C. the blots were washed threetimes with blocking buffer for 1 min each and two times for 10 min each.After air drying, the ligand binding was evaluated by autoradiography.

Isolation of p60 and p90 from rat liver membranes. Unless otherwiseindicated, all purification procedures were performed at 4° C. Thedetergent phase of the rat membrane preparation (D-phase, .sup.˜ 860 mgprotein) was applied to a polyethylenimine cellulose column (PEI) (3×30cm) equilibrated with TNE buffer. After washing with equilibrationbuffer (TNE plus 2 mM PMSF containing 1% CHAPS), the proteins bound tothe PEI column were eluted by a 240 ml linear gradient of 0.1-1.5M NaClin the equilibration buffer. Fractions were analyzed for bindingactivity by the solid-phase AGE binding assay. The active fractions werepooled and dialyzed overnight against TNE buffer containing 1% CHAPS.The PEI pool (160 mg protein, 80 ml) was then applied to aDEAE-cellulose column (2.5×20 cm) previously equilibrated withTNE/CHAPS/PMSF equilibration buffer. The column was washed with 4 columnvolumes of equilibration buffer and proteins were eluted by a 200 mllinear gradient of 0.2-1.5M NaCl in equilibration buffer. The fractionswhich contained AGE-BSA binding activity were pooled and concentrated byultrafiltration (Centricon 10, Amicon). The concentrated DEAE pool (80mg protein) was cycled three times through a BSA-Sepharose 4B column(2×12 cm, 10 mg of BSA per ml of gel), to eliminate proteins which boundto BSA. The flow-through from this BSA-Sepharose column was then appliedto an AGE-BSA-Sepharose 4B column (2×6 cm, 10 mg of AGE-BSA per ml ofgel) and cycled twice. The column was washed with 25 column volumes ofPBS buffer, pH 7.4, containing 0.2% Triton X-100, and 1 mM PMSF. Theproteins bound to the AGE-BSA column were eluted with the step-wiseaddition of PBS buffer containing 1.5M NaCl, 0.2% Triton X-100, and 1 mMPMSF. Each fraction was dialyzed against PBS containing 1 mM PMSF,concentrated by ultrafiltration (Centricon 30, Amicon) and analyzed for¹²⁵ I-AGE-BSA binding activity by the solid-phase binding assay.

Preparative electrophoresis was performed as described in detailelsewhere. In brief, 50 μg of the protein mixture which had beenaffinity purified over AGE-BSA was boiled for three min in sample buffer(0.03M Tris-HCl, pH 6.8, 1% SDS, 5% glycerol, 0.015% Bromophenol Blue)in the presence of 0.1M 2-mercaptoethanol and electrophoresed through10% polyacrylamide gels in the presence of 0.1% SDS. The 60 kD and 90 kDprotein bands were excised and electro-eluted (Elutrap, Schleicher &Schuell) in Tris buffer (25 mM, pH 8.5) containing glycine (192 mM) and0.1% SDS, as described.

Antibody generation. Laying hens were injected subcutaneously atmultiple sites with a total of 100-150 Mg of electrophoreticallypurified p60 or p90 in complete Freund's adjuvant (Pocono Rabbit Farmand Laboratory, Canadensis, Pa.). On day 14 and 21 the hens wereinjected with an additional 60 μg of each protein in complete Freund'sadjuvant. Further boosts of 80-100 μg of the corresponding proteins inincomplete Freund's adjuvant were given one month after the initialseries. Eggs and serum from the chickens immunized with p60 or p90proteins were separately collected. Immunoglobulins from the yolks wereextracted according to the method of Polson et al., while serumimmunoglobulins were isolated by a combination of ammonium sulphate(30%) precipitation and DEAE cellulose chromatography.

Western Blotting. Ten μg aliquots of detergent-solubilized samples ofmembrane protein were boiled in sample buffer in the presence of 0.1M2-mercaptoethanol, and electrophoresed through gradient gels (8-16%).After transferring onto nitrocellulose, the membranes were rinsed inTBS-t buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween 20) and blocked withTBS-t buffer containing 2% BSA for 1 hour at 4° C. The blots were probedwith either anti-60 kD or anti-90 kD avian IgG or pre-immune chicken IgG(10 μg/ml) for 60 min at 4° C. and then washed with TBS-t buffer threetimes, for 5 min each. The blots were then incubated with goatanti-chicken alkaline phosphatase conjugate (1:1000 dilution) for 1 hrat 4° C. Color development was achieved according to instructions of themanufacturer (Promega, Cleveland, Ohio).

Flow Cytometric analysis

Cell preparation. Heparinized blood was drawn from male Sprague-Dawleyrats (200-300 g) by cardiac puncture. Purified monocytes were preparedover Ficoll-Hypaque and Percoll gradients. Resident rat peritonealmacrophages were obtained from rats by washing the peritoneal cavitywith 20 ml of PBS and were characterized by flow cytometry.

Fluorescence Flow Cytometry. The expression of p60 and p90 AGE-bindingproteins on rat monocytes and macrophages was determined by indirectimmunofluorescence. Single color cell staining was performed byincubating one million cells with biotinylated anti-p60 or anti-p90primary antibodies at a final concentration of 5 μg/ml for 20 min at 4°C. Cells were washed in staining buffer (PBS, 3% FBS, 0.1% NaN₃) andthen incubated with FITC-conjugated avidin (Becton Dickinson, MountainView, Calif.). Background fluorescence was determined by staining thecells with a relevant isotypic control antibody, biotinylated chickenIgG used in identical concentrations (5 μg/ml). Cells were analyzedusing a FACSCAN (Becton Dickinson, Mountain View, Calif.) with gates setby forward angle light scatter and side scatter. Fluorescence emissionfor FITC was detected by selectively collecting at 500-537 nm on atleast 5000 labeled cells, gated to include monocytes/macrophages and toexclude lymphocytes, other non-monocytic cells and dead cells. The datawere analyzed by Paint-A-Gate software (Consort 30, Becton Dickinson,Mountain View, Calif.).

For the cross-competition study, 10⁶ rat monocytes were treated witheither 5 μg/ml biotinylated anti-p60 antibodies in the presence of20-fold excess anti-p90 antibodies, or 5 μg/20 μl biotinylated anti-p90in the presence of 20-fold excess anti-p60. The antibody-treated cellswere then labeled using FITC-avidin and analyzed by flow cytometry.

RESULTS

In vivo tissue distribution of AGE-binding activity. Applicantspreviously identified a 90 kD protein on mouse and humanmonocytes/macrophages which selectively binds AGE-proteins. Since thesesources are not convenient to provide sufficient material for furtherbiochemical characterization, alternate tissue sources were sought. As afirst step, the distribution of AGE-specific binding activity in rattissues was examined by uptake studies of ¹²⁵ I-AGE-RSA. Either ¹²⁵I-AGE-RSA (50 μg, 8.3×10⁵ cpm/μg) or ¹²⁵ I-normal RSA (50 μg, 6.2×10⁵cpm/μg) was injected intravenously into rats along with ⁵¹ Cr-labeledRBC, as described in Methods. After 10 minutes, greater than 50% of theAGE-RSA was concentrated in the liver, whereas the liver uptake ofnon-modified ¹²⁵ I-RSA was consistently less than 10% of the AGE-RSAvalues (FIG. 5A). Tissue accumulation of AGE-RSA was not affected by theprior injection of 100-fold excess non-labeled RSA (5 mg, not shown). Incontrast, pre-treatment of rats with excess non-labeled AGE-RSA (5 mg)decreased the accumulation of AGE-BSA in the liver by about 45% after10, 20 and 60 minute intervals (FIG. 5B). The uptake of AGE-RSA remaineduniformly low in all other major organs, with or without the non-labeledcompetitor. It was apparent that the liver had a high specific capacityto accumulate AGE-protein and therefore represented a potentially richsource for the isolation of the AGE-binding proteins.

1. AGE-binding assay

To facilitate the isolation of the AGE-binding proteins from liver asolid-phase assay system was developed involving the immobilization ofdetergent-solubilized membrane proteins onto nitrocellulose and probingfor ligand-specific binding activity with ¹²⁵ I-AGE-BSA as described inMethods. FIG. 6A shows the effect of increasing amounts of crude livermembrane proteins on AGE-ligand binding. Total AGE-BSA binding increasesin proportion to the amount of membrane proteins immobilized on thefilters, whereas non-specific binding was negligible. The AGE-bindingkinetics of these membrane proteins after blotting onto nitrocelluloseis shown in FIG. 6B. When .sup.˜ 8 μg of hepatic membrane proteinsimmobilized onto NC filters were incubated with increasing amounts of¹²⁵ I-AGE-BSA, saturable binding was observed with a B_(max) of 0.22pmol/8 μg of protein (FIG. 6C). A dissociation constant (K_(d) of 4×10⁻⁸M) was revealed by Scatchard analysis of the binding data.

The specificity of liver cell binding activity for AGE adducts onprotein was determined in competition experiments testing ¹²⁵ I-AGE-BSAagainst several different AGE-protein competitors, as well as againstligands known to bind to other scavenger receptors (FIG. 6D). Theaddition of 150-fold excess AGE-RNAse or AGE-collagen I completelyinhibited the binding of radio-labeled AGE-BSA to crude hepatic membraneprotein extracts immobilized on NC filters. In contrast FFI-BSA,formaldehyde-treated BSA, glucosamide-BSA (a chemically linkedglucose-BSA compound) or acetyl-LDL did not compete against the bindingof labeled AGE-BSA.

Using similar detergent-solubilized membrane preparations from heart,kidney, brain, or lung obtained by identical procedures as described forliver, ¹²⁵ I-AGE-BSA specific binding activity was examined by the samesolid-phase AGE-binding assay. Liver membrane proteins exhibited thehighest binding activity among all tissues examined, consistent with ourin vivo observations (data not shown).

2. Purification of rat liver AGE-binding proteins

Using the solid-phase AGE binding assay as a means of monitoringAGE-binding activity column fractions, the isolation of AGE-bindingprotein(s) was pursued by the procedure outlined in Table 1 anddescribed in detail in Materials and Methods. In brief, rat livermembrane proteins were solubilized in Triton X-114. After detergentphase-separation, the D-phase was subjected to chromatography onPEI-cellulose, DEAE-cellulose, BSA-Sepharose, and finally,AGE-BSA-Sepharose. After elution from the AGE-BSA Sepharose column, thefractions were assessed for AGE-binding activity by the solid-phase AGEbinding method (FIG. 7).

Analytical SDS-PAGE electrophoresis of the active fractions obtainedfrom the AGE-BSA-column revealed the presence of two main protein bandswith approximate molecular weights of 60 kD (p60) and 90 kD (p90) (FIG.7, inset). In order to separate larger amounts of these AGE-bindingproteins, AGE-BSA column eluate fractions were subjected to preparativePAGE and the individual proteins were separately electro-eluted fromrespective gel slices.

Gel-purified p60 and p90 were blotted onto Immobilon membranes and aminoterminal sequences were obtained at the Rockefeller Universitysequencing facility. Table 7 records the N-terminal sequence obtainedfrom each of these proteins, which data is also presented herein inFIGS. 11 and 12. Comparison of these sequences with the translatedGenbank database did not reveal significant similarity to other knownproteins.

Ligand blotting of p60 and p90 proteins immobilized on nitrocellulose,using ¹²⁵ I-AGE-BSA as probe, revealed that only the 60 kD protein boundthis ligand (not shown). After blotting on nitrocellulose, the 90 kDprotein did not bind ¹²⁵ I-AGE-BSA, although p90 did bind to theAGE-BSA-Sepharose matrix and was not retained on the BSA-Sepharosecolumn.

When crude rat liver membrane proteins (D-phase) were separated bySDS-PAGE under non-reducing conditions, transferred to NC filters, andprobed for ligand binding with ¹²⁵ I-AGE-BSA, a single major AGE-BSAbinding band at an approximate molecular mass of 60 kD was revealed(FIG. 8A, lane 1). The binding of this protein to AGE-BSA was inhibitedpartially in the presence of a 25-fold excess (lane 2) and completely bya 150-fold excess of non-radioactive AGE-BSA (lane 3). No otherprominent bands were observed under these conditions. It thus appearslikely that nitrocellulose immobilization may inactivate the bindingproperties of p90, or that p90 is a p60-associated protein which lacksindependent AGE-binding activity.

3. Immuno-characterization of AGE-binding proteins

Purified p60 and p90 proteins were injected into chickens in order toobtain specific polyclonal antibodies. Preparation of avian IgG specificfor each of the proteins were isolated either from egg yolk or serum asdescribed in Materials and Methods. The specificity of each of theantibodies was verified by Western blot analysis, using the same crudeliver membrane protein extract analyzed above by ligand blotting (FIG.8B). The antibody to the p60 AGE-binding protein recognized a majorprotein band at approximately 60 kD (lane 2), while the pre-immune IgGdid not (lane 1). Similarly the antibody to p90 recognized a singleprotein band at about 90 kD (lane 4), whereas the pre-immune antibodiesdid not (lane 3).

The antibodies to p60 and p90 were used to screen for the expression ofthese proteins on the surface of rat peripheral blood monocytes andperitoneal macrophages. FACS analyses which demonstrated the presence ofeach protein on the surface of both cell types are shown in FIG. 9.Panels A and B illustrate flow cytometric detection of p60 and p90 onthe surface of rat monocytes. FIG. 9 also shows that the binding ofeither anti-p60 or anti-p90 to the monocyte cell surface was notaffected by a 20-fold excess of the heterologously directed antibody(panels C and D respectively). Distinct binding of the anti-p60 andanti-p90 antibodies was also observed when the rat peritoneal residentmacrophages were analyzed by flow cytometry (FIG. 9, panels E and F,respectively). A small subgroup of highly fluorescent cells of anunspecified nature with a non-specific FITC staining pattern was alsonoted, using antibody as well as isotypic controls.

In order to confirm that p60 and p90 were both AGE-binding proteinsexpressed independently on rat peritoneal resident macrophages, ¹²⁵I-AGE-BSA binding inhibition experiments were carried out using eachantibody separately (undiluted: 10 μg/ml) as well as in combination(FIG. 10A). In the presence of increasing concentrations of anti-p60antibody significant AGE-BSA binding inhibition was observed (up to 80%at a final concentration of 10 μg/ml). Similarly ¹²⁵ I-AGE-BSA bindingwas inhibited up to 60% by the anti-p90 antibody, while the combinationof both antibodies at a dilution of 1:10 provided 84% inhibition. Whenradio-labeled FFI-BSA (made from the chemically synthesized model AGEcompound, FFI, crosslinked onto BSA with a carbodiimide reagent) wasused as the ligand, anti-p60 as well as anti-p90 mediated aconcentration-dependent inhibition of FFI-modified BSA binding (FIG.10B). Moreover, and as found with AGE-BSA, a combination of anti-p60 andanti-p90 antibodies exerted greater inhibition compared to eitheranti-p60 alone or anti-p90 alone. No inhibitory effect was noted whenisotypic control antibodies were used, even at the maximalconcentration, in conjunction with either modified BSA ligand (notshown).

                                      TABLE I                                     __________________________________________________________________________    Purification of Rat Liver AGE Binding Proteins                                          .sup.125 I-AGE-BSA Binding                                          Purification                                                                            Total Total                                                                             Specific Activity                                                                     Purification                                                                        Recovery                                    Step      Protein (mg)                                                                        μg                                                                             (μg/mg)                                                                            factor                                                                              (%)                                         __________________________________________________________________________    Liver Membrane                                                                          6,800 455.5                                                                             0.067   1     100                                         D-phase   830   207.5                                                                             0.256   3.7   45                                          PEI       175   262.2                                                                             1.498   22    58                                          DEAE      82    153.5                                                                             7.490   109   34                                          AGE-affinity                                                                            0.560 92.4                                                                              165     2463  20                                          Preparative PAGE*                                                                       0.180 --  --      --    --                                          __________________________________________________________________________     Rat liver membrane proteins were procured and prepared as described.          Specific binding of AGEBSA was determined by the solid phase binding          assay.                                                                        *Binding activity could not be determined due to the presence of SDS.    

                  TABLE II                                                        ______________________________________                                        N-Terminal Amino Acid Sequence Analysis of                                    AGE-Binding Proteins from Rat Liver                                           ______________________________________                                        60 kD:       XGPRTLVLLDNLNVRDTHXLFF                                           90 kD:       XEVKLPDMVSLXD                                                    ______________________________________                                         X INDICATES UNIDENTIFIED RESIDUES.                                       

DISCUSSION

The above experiments confirm the isolation and discovery of two novelrat liver membrane proteins, designated p60 and p90 by their migrationSDS-PAGE, which specifically bind to protein ligands modified byadvanced glycosylation endproducts (AGEs). Amino terminal sequenceanalysis indicates that these proteins bear no significant homology toeach other nor to any previously sequenced proteins currently availablein the Genbank database. Both p60 and p90 are present on rat monocytesand macrophages and are immunoreactively and functionally distinct. Ofimportance is the fact that these binding proteins have beendistinguished from both the recently reported macrophage scavengerreceptor for acetyl-LDL, a functional trimer composed of three 77 kDglycoprotein subunits; and from the binding proteins forformaldehyde-treated albumin with M_(r) 's of 30 and 52 kD.

The p60 and p90 AGE-binding proteins were isolated from rat liver, uponthe determination that this organ acts as a major filter for the in vivoclearance of AGE-modified macromolecules; liver presents the highestcapacity to specifically sequester AGE-proteins administeredintravenously (FIG. 5). An isolation procedure, including elution ofdetergent-solubilized membrane proteins from an AGE-protein affinitymatrix was devised, and the p60 and p90 AGE-binding proteins were foundto co-purify. When immobilized on nitrocellulose, however, only p60retained binding activity for AGE-modified ligands. Just as theyco-purify over anion exchange and ligand affinity columns, p60 and p90were also observed to co-purify in hydroxylapatite chromatography (notshown). The extent of this co-purification continues to be examined.

The liver is a complex organ containing several cell types, includingmacrophages and endothelial cells, both of which have been shown to bearAGE-receptors. To determine whether macrophages also expressed a 60 kDAGE-binding protein, and whether there was any relationship between themacrophage 90 kD and the liver p90 AGE-binding proteins, specificpolyclonal antibodies to liver p60 and p90 were developed.

The specificity of these antibodies was demonstrated by Western analysisof crude liver membrane extracts, revealing that each antiserumidentified a single protein band of the appropriate molecular weight.Flow cytometric analysis of rat peripheral monocytes and peritonealresident macrophages revealed that each antisera bound to the surface ofboth of these cell types. Cross-competition studies performed onmonocytes revealed no cross-reactivity between the two antibodies. Thesedata indicate that the p60 and p90 AGE-binding molecules originallyisolated from whole liver preparations are each present on monocytes aswell as macrophages (FIG. 9).

Flow cytometric binding inhibition experiments clearly demonstrated thatp60 and p90, expressed on the surface of monocytes/macrophages,independently bound AGE-modified ligands. Interestingly, a combinationof antibodies specific for p60 and p90 mediated greater inhibition ofAGE-protein binding than did either antibody alone.

Either antiserum, used independently or in combination, prevented morethan 90% of binding FFI-BSA to rat macrophages. In the case of p60, thisfinding is surprising given that this binding protein does not bindFFI-BSA in a solid-phase ligand blotting assay. With regard to liverp90, the flow cytometry and FFI-binding inhibitory data indicate thatthis molecule may be closely related to the 90 kD protein isolated frommurine RAW 264.7 cells. In fact, preliminary experiments usingantibodies raised against rat liver p60 and p90 proteins to stain mouseRAW cells provided flow cytometric and binding inhibition resultssimilar to those obtained with rat monocytes and macrophages, stronglysupporting a structural similarity between the AGE-binding proteins ofthese two rodent species.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Rat                                                             (G) CELL TYPE: Liver membrane                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 1..13                                                           (C) OTHER INFORMATION: Xaa denotes unidentified residues                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       XaaGluValLysLeuProAspMetValSerLeuXaaAsp                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Rat                                                             (B) CELL TYPE: Liver Membrane                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 1..23                                                           (C) OTHER INFORMATION: Xaa denotes unidentified residues                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       XaaGlyProArgThrLeuValLeuLeuAspAsnLeuAsnValArg                                 151015                                                                        ArgAspThrHisXaaLeuPhePhe                                                      20                                                                            __________________________________________________________________________

What is claimed is:
 1. An isolated antibody that binds a receptorprotein, said receptor protein to which said antibody is raisedcomprising a protein in purified form derived from rat liver cellmembranes, that recognizes and binds advanced glycosylation endproducts(AGE) and that possesses the following characteristics:A. said proteinrecognizes and binds with the ligands AGE-RNase and AGE-Collagen I; B.said protein does not recognize and bind with the ligands FFI-BSA,formaldehyde-treated BSA, glucosamide-BSA, and acetyl LDL-BSA in solidphase ligand blotting assay; and C. said protein has a molecular mass ofabout 90 kD or about 60 kD as determined by protein migration onSDS-PAGE.
 2. The antibody of claim 1 wherein said protein has thefollowing characteristics:A. said protein is found on rat liver cellmembranes; B. said protein is expressed on rat monocytes and ratmacrophages; and C. said protein is purified by elutions based,respectively, on an AGE ligand affinity column, and anion exchangecolumn, and a hydroxylapatite column;and also bears the followingdistinction: D. when said protein has a molecular mass of about 90 kD,as determined by SDS Page, and it is immobilized on nitrocellulose in asolid phase assay, the 90 kD protein does not bind to AGE-modifiedligands, and when the protein has a molecular mass of about 60 kD, asdetermined by SDS Page, said protein binds to AGE modified ligands whenimmobilized on nitrocellulose in a solid phase assay.
 3. The antibody ofclaim 1 wherein said receptor is isolated and purified by theimmobilization of detergent-solubilized rat liver membrane proteins onnitrocellulose in accordance with a solid phase assay protocol.
 4. Theantibody of claim 1 wherein the said protein as determined by SDS Pageshas a molecular mass of about 90 kD, and the NH₂ -terminal partial aminoacid sequence set forth in FIG. 11 (SEQ ID NO.2).
 5. The antibody ofclaim 1 whrein the said protein as determined by SDS Page has amolecular mass of about 60 kD and the NH₂ -terminal partial amino acidsequence set forth in FIG. 12 (SEQ ID NO.1).
 6. The antibody of claim 1which is a polyclonal antibody.
 7. The antibody of claim 1 which is amonoclonal antibody.
 8. An immortal cell line that produces a monoclonalantibody according to claim
 7. 9. The antibody of claim 1 labeled with adetectable label.
 10. The antibody of claim 9 wherein the label isselected from the group consisting of enzymes, chemicals which fluoresceand radioactive elements.